The aim of Project 1A is to develop a molecular genetic approach to extend the analysis of learned fear to the molecular level. This approach involves two phases: (1) discovery of genes important for learned fear, and (2) mechanistic studies of gene candidates and the development of animal models of anxiety. In a large sense, we hope that in accomplishing phases (1) and (2), we will be able to contribute to developing a paradigm for obtaining genetic insight into a complex mental disorder: the spectrum of Anxiety Disorders. In phase 1 of Project 1A we propose to study genes induced in the amygdala during learned fear, a form of fear that engages the neural mechanisms that are driven by danger and threat, and which sets in place long-term and enduring adaptive defensive responses. It is a naturally occurring and highly adaptive function of the brain, and it has proved to be experimentally tractable at the level of behavior, anatomy, information processing, and molecular genetics, and each of these levels is reasonably conserved across species. By using fear conditioning, we know we are activating fundamental and phylogenetically ancient mechanisms that support appropriate emotional responses to danger. Since anxiety disorders all involve inappropriate fear responses, we are hopeful that by identifying the molecular genetic basis of fear conditioning we will uncover means of probing the genetic mutations that underlie anxiety disorders. To maximize our identification of candidate genes, we will drive the defense system with a wide range of fear-conditioning methodologies, using both 1st and 2nd order cued conditioning, 1st and 2nd order unpaired cue conditioning (conditioned inhibition of fear), and contextual conditioning. All of these behavioral protocols have been fully developed in our hands. In Phase 2 of Project 1A, we will screen for genes that are regulated in the amygdala of wild-type mice exposed to learned fear, and then developing genetically engineered mice that have trans genes regionally restricted to the amygdala and temporally regulated. In addition, we will study, in a like manner, other genes that are known to be important for learned fear. Our mechanistic studies will involve behavioral, electrophysiological, and molecular genetic studies designed to examine the effects of genes identified in the gene discovery phase on behavioral fear conditioning and on amygdala function. In doing so, we will attempt to relate these genes to signal transduction pathway important for LTP on the one hand and to behavioral learning of fears, safety, context and 2nd order learning on the other. We have already succeeded in expressing transgenes in the lateral nucleus of the amygdala, including a Grp-IRES-tTA knock-in cassette. This mouse line shows tTA expression at high levels in the lateral nucleus; this nucleus is known to be an input for both US and CS for Pavlovian fear-conditioning. One of the genes so identified is GRP, and its receptor, GRPR. We have examined mice with knockout of GRPR and found a powerful enhancement of fear. We continue to work with single cell cDNA libraries from the lateral amygdala to delineate other promoters that generate restricted patterns of expression. However, we would emphasize that even though we will be able to express genes at high levels in the lateral nucleus, this expression is not likely to be completely restricted and this has so far proved to be unavoidable.